Abstract Immune checkpoint inhibitors have recently emerged as a revolution in cancer care, providing the potential for durable response and improved survival for numerous cancer patients across multiple cancer types. However, only 10-20% of patients who are treated with checkpoint inhibitors have responded in most cancer types, necessitating biomarker assays that can reliably and accurately identify those patients likely to respond. Recent, compelling evidence supports the clinical utility of microsatellite instability (MSI) as a predictive marker of immunotherapy response, reaching overall survival rates of >64% versus 11% in microsatellite stable (MSS) tumors. MSI-high tumors are histopathologically distinct from their MSS counterparts, displaying a marked influx of tumor-infiltrating lymphocytes and express high levels of immune checkpoint inhibitors, including PD-1. This unique immune microenvironment supports the hypothesis that MSI-high tumors more susceptible to immunotherapies, and has been proposed as a biomarker for stratifying patient populations. Supporting this notion, The FDA just granted priority review for a supplemental application of Keytruda (a PD-1 inhibitor) in previously treated MSI-H tumors on the basis of 5 open-label Phase I/II trials across multiple tumor types. Current MSI screening is conducted on tumor biopsies using variable, non- standardized PCR-based and IHC laboratory developed tests (LDTs), creating the need for a standardized, accurate, non-invasive assay that may be decentralized to inform the clinical management of patients for treatment with immunotherapies. Cell-free tumor-specific genetic alterations, or circulating tumor DNA (ctDNA), are detectable in the plasma of ?80% of cancer patients with metastatic disease. Reliable detection of tumor specific alterations in plasma can aid in the stratification of patients for targeted therapies and overcome the need for tumor tissue by traditional molecular approaches. The goal of this proposal is to develop a stand-alone molecular diagnostic assay to determine MSI status from plasma in cancer patients (known as CancerPRO MSI) to guide immunotherapy for multiple indications.